Early Diagnosis of HIV-1 and HIV-2 Using Cobas HIV-1/HIV-2 Qualitative Test

A Novel Qualitative Nucleic Acid Amplification Test for Plasma, Serum, and Dried Blood Spot Specimens

Lucia Hans, MB BCh, FCPath; Nicole von Allmen, PhD; Anke Edelmann, PhD; Jörg Hofmann, PhD; Alex Y. Nilsson, PhD; Christian O. Simon, PhD; Britta Seiverth, PhD; Peter Gohl, PhD; Sergio Carmona, PhD

Disclosures

J Acquir Immune Defic Syndr. 2021;87(5):1187-1195. 

In This Article

Methods

Study Procedures and Description of Device

This multicenter evaluation was conducted in Germany (Berlin and Ingelheim) and at National Health Laboratory Services, Johannesburg, South Africa. The protocol received ethical approval from the Ethikkommission der Universitätsmedizin Charité, Berlin (EA1/177/17), and the University of the Witwatersrand Human Research Ethics Committee (M150160). All specimens were unlinked and anonymized, and the study results were not used for patient management.

The cobas HIV-1/2 Qual test combines automated nucleic acid extraction and purification, with real-time PCR and result reporting separately for HIV-1 and HIV-2. It targets the HIV-1 long-terminal repeat and gag regions and HIV-2 long-terminal repeat region. For DBS testing, the assay required 70 μL of whole blood; spots were removed from the specimen collection card using disposable tweezers and transferred to Greiner Cryo.s tube (Figure 1). Then, 1150 μL of cobas specimen preextraction reagent was added to each tube, which was placed in an Eppendorf Thermomixer and incubated for 10 minutes at 56°C and 1000 rpm. After incubation, the tubes were decapped, loaded, and processed on the cobas 6800/8800 instrument, together with tubes containing plasma and serum samples (650 μL volume in each). Handling of instruments, specimens, controls, and reagents was performed according to procedures described in the cobas 6800/8800 User Guide (version 3.0, Software version 1.2).

Figure 1.

Overview of the cobas HIV-1/2 Qual test workflow for DBS.

Method Correlation and Confirmation of HIV Infection

Four assessments were performed to validate the technical performance of the cobas HIV-1/2 Qual test. Assessments in adults were performed using leftover plasma and serum specimens from HIV-infected patients. Pediatric assay performance was evaluated using remnant DBS samples. Only samples with valid results for both the test under evaluation and the comparator tests were included in each evaluation (differences between the number of tests performed and results reported were due to invalid runs or samples). Discrepant samples were tested with heminested PCR and post-PCR ultraperformance liquid chromatography. These tests assisted in determining whether an observed signal was a true-positive result or a nonspecific amplification event. In addition, Elecsys HIV combi PT fourth-generation test (Roche Diagnostics GmbH, Penzberg, Germany) was used to confirm HIV-1–negative results in case of discordance, and sequencing analysis was used to confirm HIV-2–negative results.

The cobas HIV-1/2 Qual test results of plasma and serum samples (n = 339) were compared with those of the recomLine HIV-1 & HIV-2 Immunoglobulin G (IgG) (Mikrogen GmbH, Neuried, Germany), a CE-marked serological test that differentiates between HIV-1 and HIV-2. Plasma samples (n = 150) were compared with those on COBAS AmpliPrep/COBAS TaqMan (CAP/CTM), a CE-marked PCR test for HIV-1. Specimens were analyzed in single determinations. In addition, to assess the ability to detect HIV in patients who had not received antiretroviral treatment, 30 plasma and 30 serum samples from untreated patients with confirmed positive results for HIV-1 antigen and antibody were tested with the cobas HIV-1/2 Qual test.

Performance of cobas HIV-1/2 Qual test in early infant diagnosis was assessed against CAP/CTM using 311 DBSs from children aged 18 months or younger born to HIV-positive mothers. Samples were spotted onto Munktell TFN cards (n = 283) or Whatman 903 cards (n = 28).

Specificity

Specificity of cobas HIV-1/2 Qual test was determined by testing HIV-1/-2–negative plasma (n = 613), serum (n = 607), and DBS (n = 604) samples. Samples were collected from HIV-negative volunteers.

Genotype Inclusivity

To confirm genotype inclusivity of the cobas HIV-1/-2 Qual test, reference panels representing different HIV-1 and HIV-2 subtypes were analyzed. These panels consisted of HIV-1 group M subtypes A, C, D, F, G, H, J, and K and the circulating recombinant forms CRF01_AE, CRF02_AG, CRF12_BF, and CRF14_BG. Samples from HIV-1 groups N and O and HIV-2 groups A and B were also included. All specimens were previously confirmed to be HIV-positive with licensed serological tests and/or NATs and had HIV viral load levels commonly seen in infected patients. The reactivity of each target was determined in undiluted samples and in samples diluted in HIV-negative pooled plasma or serum to near the limit of detection (LOD) of the assay. For most genotypes, 10 panels were tested, but, because of limited availability, fewer panels were tested for HIV-1 group M subtypes J (5) and K (9), circulating recombinant forms CRF12_BF (2) and CRF14_BG (9), and HIV-1 group N (1).

Limits of Detection

Three independent dilution series were prepared consisting of 6 concentration levels for HIV-1 groups M and O and 5 concentration levels for HIV-2 (Table 4). The individual intermediate stock solution aliquots were diluted in HIV-negative pooled plasma and serum. Dilution series were also prepared for DBSs in whole blood of 3 independent clinical samples.

Each panel was tested over multiple days, operators, systems, reagent lots, runs, and replicates per run. In total, with plasma and serum samples, 63 replicates per concentration level were tested for HIV-1 groups M and O, 42 replicates per concentration level were tested for HIV-2, and 84 DBS replicates per concentration level were tested for HIV-1 group M and HIV-2. For each target, the LOD was based on the probit value at the 95% hit rate, using the combined data from all lots. In addition, we determined the lowest concentration level with a ≥95% hit rate and the percentage of detection at 50% LOD using probit analysis.

Performance on Seroconversion Panels

We evaluated 35 HIV-1 group M commercially available seroconversion panels obtained from Zeptometrix, Inc. (Buffalo, NY) and Boston Biomedica, Inc-SeraCare Diagnostics (West Bridgewater, MA), each with a certificate of analysis. A single replicate was tested undiluted using the cobas HIV-1/2 Qual test. Three assessments were performed. The results of cobas HIV-1/2 Qual test were compared with the findings of a qualitative confirmatory assay for detecting antibodies to HIV-1 and HIV-2 (Bio-Rad Geenius HIV 1/2 Confirmatory Assay; Bio-Rad, Marnes-la-Coquette, France), a fourth-generation HIV immunoassay (Abbott ARCHITECT HIV Ag/Ab Combo test; Abbott, Wiesbaden, Germany), and a NAT (cobas TaqScreen MPX, v2.0). We reported the mean days to the first positive results and the difference in number of days to the detection between the cobas HIV-1/2 Qual test and the other assays.

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